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Effects of hydrogen peroxide on slow- and fast-growing NIH/3T3-derived cultures: nuclear and cytoplasmic aspects related to senescence and transformation
No full textCellular senescence can occur with similar phenotypes in normal cells, during aging, and in tumor cells, spontaneously or after cytostasis. The fall or increase in proliferative activity are key aspects of the respective conditions, in which the levels of reactive oxygen species can vary, affecting the cellular redox homeostasis. This work aimed to study the relationships between senescence and transformation by comparing cells with different proliferative activities and phenotypes attributable to transformation (NIHs cultures) or senescence (NIHv cultures), before and after incubation with hydrogen peroxide. Both cultures were derived from the NIH/3T3 cell line, which was used here as a reference (NIHb), after the serum starvation. Our experimental model can be representative of the heterogeneity of cell subpopulations, with different degrees of transformation and senescence, found in some tumors. The characterization of the functional properties of NIHb, NIHs, and NIHv cells was performed by a morphocytometric analysis of the cell cycle progression, mitochondrial and lysosomal content/activity, and superoxide anion production. The efficiency of the lysosomal compartment was also assessed by estimating the autophagic activity and measuring lipofuscin autofluorescence. Comparisons of nuclear and cytoplasmic parameters before and after the incubation with hydrogen peroxide revealed differences in the expression and modulation of cellular senescence patterns. The treatment effects were very limited in the NIHb culture; the senescence condition was essentially maintained in the NIHv cells, while the most relevant changes were found in the NIHs cells. In the latter, the acquisition of the senescent phenotype, also demonstrated by the positivity of SA-β-galactosidase, was correlated with a decrease in proliferative activity and a change in the content/activity of the mitochondria and lysosomes, which showed similarities with the basal senescence conditions of NIHv cells. In NIHs cells, increased autophagy events and lipofuscin accumulation also indicate the establishment of cytoplasmic dynamics typical of senescence. The variable responses to hydrogen peroxide, besides depending on the different basal cytokinetic activity of the cultures examined, appeared to be related to the specific cell redox state resulting from the balance between endogenous ROS and those produced after treatment. Especially in NIHs cells, the slowing down of the cell cycle was linked to dynamic interconnections between the mitochondrial and lysosomal compartments. This would indicate that transformed cells, such as NIHs, may express morpho-functional aspects and markers typical of cellular senescence, as a consequence of the modulation of their redox state
Polyploid cell dynamics and death before and after PEG-treatment of a NIH/3T3 derived culture: vinblastine effects on the regulation of cell subpopulations heterogeneity
No full textBackground: Neoplastic subpopulations can include polyploid cells that can be involved in tumor evolution and recurrence. Their origin can be traced back to the tumor microenvironment or chemotherapeutic treatment, which can alter cell division or favor cell fusion, generating multinucleated cells. Their progeny, frequently genetically unstable, can result in new aggressive and more resistant to chemotherapy subpopulations. In our work, we used NIHs cells, previously derived from the NIH/3T3 line after serum deprivation, that induced a polyploidization increase with the appearance of cells with DNA content ranging from 4 to 24c. This study aimed to analyze the cellular dynamics of NIHs culture subpopulations before and after treatment with the fusogenic agent polyethylene glycol (PEG), which allowed us to obtain new giant polyploid cells. Successively, PEG-untreated and PEG-treated cultures were incubated with the antimicrotubular poison vinblastine. The dynamics of appearance, decrease and loss of cell subpopulations were evaluated by correlating cell DNA content to mono-multinuclearity resulting from cell fusion and division process alteration and to the peculiarities of cell death events.
Results: DNA microfluorimetry and morphological techniques (phase contrast, fluorescence and TEM microscopies) indicated that PEG treatment induced a 4-24c cell increase and the appearance of new giant elements (64-140c DNA content). Ultrastructural analysis and autophagosomal-lysosomal compartment fluorochromization, which allowed us to correlate cytoplasmic changes to death events, indicated that cell depletion occurred through distinct mechanisms: apoptotic death involved 2c, 4c and 8c cells, while autophagic-like death involved intermediate 12-24c cells, showing nuclear (lobulation/micronucleation) and autophagic cytoplasm alterations. Death, spontaneously occurring, especially in intermediate-sized cells, was increased after vinblastine treatment. No evident cell loss by death events was detected in the 64-140c range.
Conclusions: PEG-treated NIHs cultures can represent a model of heterogeneous subpopulations originating from cell fusion and division process anomalies. Altogether, our results suggest that the different cell dynamics of NIHs subpopulations can affect the variability of responses to stimuli able to induce cell degeneration and death. Apoptptic, autophagic or hybrid forms of cell death can also depend on the DNA content and ability to progress through the cell cycle, which may influence the persistence and fate of polyploid cell descendants, also concerning chemotherapeutic agent action
Static cytofluorometry and fluorescence morphology of mitochondria and DNA in proliferating fibroblasts
No full textChanges on lysosomal compartment during PMA-induced differentiation of THP-1 monocytic cells: Influence of type I and type IV collagens
No full text""hesion during phorbol-12-myristate-13-acetate (PMA)- induced differentiation of THP-1 monocytic cell line was studied. In particular, by morphocytochemical and cytometric approaches, the influence of type I and type IV collagens in an experimental model rep- resentative of three phases (initial, intermediate and terminal) of monocyte-macrophage transition was analyzed. The cells in these three phases of differen- tiation were obtained by using 6, 30 e 60 nM PMA. In this experimental model, referring to adhesion to glass as control, by using the azo-dye coupling me- thod, we have considered the analysis of Acid Phos- phatase (AcP) activity as a marker of differentiated status expression, in relation to the acquisition of macrophagic phenotype. Endosomal\\\/lysosomal sys- tem was further characterized by taking in account the uptake of fluorescent probe LysoTracker Red. Fluorochromization in the various experimental con- ditions was analyzed morphologically (fluorescence microscopy) and quantitatively (static cytometry). Data related to lysosome compartment were integra- ted, from a cytokinetic point of view, by flow cy- tometry measurements of DNA\\\/protein content. Our results have indicated that type I and type IV colla- gens were able to influence, with respect to glass ad- hesion, various differentiation phases. Type I collagen showed the higher effects in the condition of high dif-ferentiation (60 nM PMA), causing an increase in AcP activity and lysosomal system. Type IV collagen, besides determining effects on lysosomal compart- ment of intermediate and terminally differentiated cells, influenced mainly proliferative activity of cells with initial differentiation level (6 nM PMA)."
Relationship between actin microfilaments and plasma membrane changes during apoptosis of neoplastic cell lines in different culture conditions
Get PDFIn this study we investigated the relationship between the reorganisation of actin cytoskeleton and the changes at cell surface level (i.e. PS exposure and blebbing) in two neoplastic cell lines during apoptosis: Chang liver cells (adherent culture) and promyelocytic HL-60 cells (suspension culture), treated with the podophyllotoxin derivative VP16. The morphological analysis, performed by means of conventional fluorescence microscopy and confocal laser scanning microscopy, on Chang cells showed that onset and progress of the two processes are synchronised. The initial disassembly of stress fibers was associated with the early PS exposure on the cell surface. Moreover, the accumulation of actin at cortical level appeared strongly associated with an intense labelling for Annexin V and, in some cases, especially in the areas of membrane blebbing. The double staining for actin and PS exposure, quantitatively analysed by flow cytometry in HL-60 cells after different treatment times, demonstrated that the decrease of Annexin V binding in the late stages of apoptosis is associated with the strong reduction of actin labelling probably also due to a proteolytic cleavage. These events were also partially related to variations of 255 the functional state of mitochondria, by analysing cytofluorometrically the dissipation of the inner membrane potential (DYm)
Relationship between actin microfilaments and plasma membrane changes during apoptosis of neoplastic cell lines in different culture conditions
No full textDifferent expression of apoptosis during monocyte/macrophage differentiation induced by PMA.
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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